A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Remy, Ingrid; Michnick, Stephen W.

doi:10.1038/nmeth979

Cited by 399 publications

(411 citation statements)

References 15 publications

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“…Ideally, the confirmatory method can also be performed in vivo, in intact plant tissue. Examples of such techniques are FRET-FLIM and splitluciferase assays (Remy and Michnick, 2006;Bayle et al, 2008;Gehl et al, 2011). If the protein-protein interaction results in a subcellular translocation of at least one of the proteins, coexpression and translocation assays can also be applied (Piljić and Schultz, 2008;Schlü cking et al, 2013;Offenborn et al, 2015).…”

Section: Validation By Independent Methodsmentioning

confidence: 99%

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

2016

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ORCID ID: 0000-0001-7502-6940 (R.B.)Techniques to detect and verify interactions between proteins in vivo have become invaluable tools in functional genomic research. While many of the initially developed interaction assays (e.g., yeast two-hybrid system and split-ubiquitin assay) usually are conducted in heterologous systems, assays relying on bimolecular fluorescence complementation (BiFC; also referred to as split-YFP assays) are applicable to the analysis of protein-protein interactions in most native systems, including plant cells. Like all protein-protein interaction assays, BiFC can produce false positive and false negative results. The purpose of this commentary is to (1) highlight shortcomings of and potential pitfalls in BiFC assays, (2) provide guidelines for avoiding artifactual interactions, and (3) suggest suitable approaches to scrutinize potential interactions and validate them by independent methods.

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Section: Validation By Independent Methodsmentioning

confidence: 99%

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

2016

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“…To study the effects of PREP in the earliest stages of the aSyn aggregation process, we first developed an assay for studying aSyn oligomer formation in live cells. This system is based on PCA where complementary fragments of G. princeps luciferase (GLuc) are attached to interacting proteins of interest and a luminescence signal is generated upon protein interaction in live cells (31). We first expressed the aSyn-GLuc1 and aSyn-GLuc2 PCA reporter constructs in cells together with wild-type PREP and PREP(S554A).…”

Section: Prep Overexpression Increases Asyn Reporter Dimerizationmentioning

confidence: 99%

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Savolainen

Myöhänen

et al. 2015

Journal of Biological Chemistry

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Background: Prolyl oligopeptidase (PREP) modulates accumulation of aggregated ␣-synuclein both in vitro and in vivo, but the mechanism remains unknown. Results: Using live-cell and cell-free methods, we show that PREP interacts directly with ␣-synuclein. Conclusion: PREP binds to ␣-synuclein and enhances its dimerization. Significance: These results establish a mechanism of how PREP inhibition reduces ␣-synuclein aggregation and support PREP inhibition as a novel therapy in synucleinopathies.

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“…To further test a direct interaction between AR and LXR, we employed a Gaussia luciferase complementation assay (26). The two putative interaction partners are fused to different halves of Gaussia luciferase (either the N-terminal hGluc(1) or C-terminal hGluc(2)).…”

Section: The Ar Does Not Influence Lxr Expression or Lxr-dnamentioning

confidence: 99%

Cross-talk between the Androgen Receptor and the Liver X Receptor

Krycer¹,

Brown

2011

Journal of Biological Chemistry

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High cholesterol levels are associated with prostate cancer development. Androgens promote cholesterol accumulation by activating the sterol-regulatory element-binding protein isoform 2 (SREBP-2) transcription factor. However, SREBP-2 is in balance with the liver X receptor (LXR; NR1H2/NR1H3), a transcription factor that prevents cholesterol accumulation. Here, we show that LXR activity is down-regulated by the androgen receptor (AR; NR3C4). In turn, this reduces LXR target gene expression. This antagonism on LXR is also exerted by other steroid hormone receptors, including the estrogen, glucocorticoid, and progesterone receptors. This suggests a generalizable mechanism, but the AR does not affect LXR mRNA levels, protein degradation, or DNA binding. We also found that the AR does not require protein synthesis to influence LXR, suggesting a direct antagonism. However, the AR does not directly bind LXR. The AR N-terminal domain (involved in transactivation), but not its DNA-binding domain, is required to suppress LXR activity, suggesting coactivator competition. Overall, this androgen-mediated antagonism of LXR complements SREBP-2 activation, providing a more complete picture as to how androgens increase cellular cholesterol levels in a prostate cancer setting. Given the cross-talk between other steroid hormone receptors and LXR, hormonal regulation of cholesterol via LXR may occur in a variety of cellular contexts.

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A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Cited by 399 publications

References 15 publications

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Cross-talk between the Androgen Receptor and the Liver X Receptor

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